A nucleic acid array consists of a carrier on which many nucleic acid probes (hereinafter sometimes referred to as “probes”) are independently immobilized at high density without being mixed. A probe immobilized on a nucleic acid array functions as a sensor for capturing a nucleic acid molecule consisting of a sequence complementary to the nucleotide sequence of the probe by means of hybridization.
Conventionally, a product consisting of a carrier made of surface-treated glass, silicone or the like on which probes are immobilized is utilized as a nucleic acid array. Recently, such carriers have been modified using other techniques such as a gel carrier.
As methods for producing a nucleic acid array, a method in which nucleic acid probes prepared in advance are immobilized on a substrate such as slide glass and silicone, and a method in which nucleic acid probes are directly synthesized on a substrate are known.
For example, according to the optical lithography method in which probes are directly synthesized on a substrate, a nucleic acid array can be prepared by using a substance having a protective group which is selectively removed by light irradiation, combining the photolithography technique with the solid-phase synthesis technique, and by selectively synthesizing (masking) DNA on a predetermined region (reaction site) of a tiny matrix (Science 251, 767-773 (1991)).
A typical example of a method for immobilizing probes prepared in advance is a spotting method (Science 270, 467-470 (1995)). According to this method, drops of a solution comprising probes prepared by PCR or artificial synthesis in advance, which have a tiny volume of several nanoliters (n1) to several picoliters (p1), are arrayed on the surface of a chip using a particular apparatus (a spotter an arrayer), and thereby the probes are immobilized on a specific region of the substrate. In addition to the above-described method, a method for producing a microarray using a hollow-fiber-arranged body has been developed. According to this method, a base having through-holes is produced using a hollow-fiber-arranged body in which a plurality of hollow fibers made of synthetic polymer are regularly arrayed in the direction of the fiber axis. One of the features of this production method is that a lot of microarray products having the same specification can be produced from the same rod by immobilizing probes in the hollow portion of each of the hollow fibers in the hollow-fiber-arranged body and by slicing the hollow-fiber-arranged body in the direction perpendicular to the direction of the fiber axis (Japanese Patent No. 3488456).
In a nucleic acid detection method utilizing a nucleic acid array, nucleic acid samples targeted for a test are sequence-specifically hybridized to probes immobilized on the nucleic acid array, and sequence-specifically formed hybrids are detected using a fluorescent substance or the like. According to this method, nucleic acid molecules comprising nucleotide sequences in the samples, which correspond to a plurality of probes, can be examined quantitatively or qualitatively. Therefore, the method is used for analyzing the expression amount of a plurality of nucleotide sequences or the sequence itself of a specific nucleotide sequence.
Usually, in the above-described nucleic acid detection, a hybridization reaction is caused under appropriate preset conditions, and nucleic acid samples and other unnecessary substances remaining on the surface of the array are removed by washing to detect nucleic acid samples forming specific hybrids with probes. A probe is often designed to be complementary or identical to a nucleotide sequence desired to be detected and used for the purpose of sequence analysis, function analysis or the like. As a probe, a nucleic acid having a relatively long chain such as cDNA or the like, a synthetic oligonucleic acid having a relatively short chain, or the like is used. In the case where a synthetic oligonucleic acid is used as the probe for detecting a nucleic acid of human, mouse or other biological organisms, for which the findings of gene information are accumulated, the nucleotide sequence information thereof is usable. Using such nucleotide sequence information, and in consideration of the homology, function and the like of each of such sequences, the sequence of a synthetic oligonucleic acid is designed. Thus, a probe can be produced.
Such nucleic acid arrays can be provided for gene analysis (gene expression, gene polymorphism and the like), can be utilized for research applications such as the discovery of the mechanism of life phenomenon, diagnosis/therapy of diseases and the like, and are further expected to be applied to industrial applications such as breed classification made by differentiating the gene type and the like. In the meantime, in order to apply such nucleic acid arrays to industrial applications, it is essential to maintain the quality thereof. Therefore, one urgent need is to establish a quality control method for guarantee of quality.
Among quality control items, the most important task is to examine whether or not probes immobilized on a nucleic acid array produced are accurately immobilized on predetermined positions. As an example of a method of the above-described examination, a method, in which a nucleic acid array is immersed in a nucleic acid staining agent such as ethidium bromide to stain a probe or a predetermined position on which a probe is immobilized, is known. According to this method, the presence or absence of the probe on/in the nucleic acid array can be known, but it cannot be examined whether or not the probe is immobilized on the predetermined position. Moreover, when utilizing the stained nucleic acid array as it is for a test or the like, the staining agent is a noise in the detection. Therefore, in order to provide nucleic acid arrays as products, it is necessary to conduct an operation, in which a stain (ethidium bromide) that stains probes or predetermined positions on which probes are immobilized must be completely washed away. This procedure must be performed on a product-by-product basis. Therefore, this is a very complicated procedure.
Moreover, in order to confirm spot positions, it is necessary to prepare labeled nucleic acids, which correspond to all probes immobilized on an array, as complementary chains of the probes, and to conduct a detection operation by means of hybridization on a probe to probe basis. When a large number of probes are immobilized, operations are more complicated and unpractical.
Thus, it is very important to easily confirm “what kind of sequence a probe has and on which position of a nucleic acid array the probe is immobilized” to guarantee the quality of the nucleic acid array.
However, presently almost no operation for confirmation is performed. That is because, as described above, since a nucleic acid array has a lot of probes immobilized on a carrier, operations for confirmation are complicated. Moreover, that is because it is difficult to easily differentiate sequences of probes themselves.
That is, in order to confirm what kind of probe is present on which position of a nucleic acid array once prepared, only the information obtained from the production process of the nucleic acid array can be relied on. It is extremely difficult to determine each position on which each probe is immobilized after the production.
If unexpected probes are immobilized on unexpected positions of a nucleic acid array, with respect to probes whose immobilized positions are wrong, wrong data may be submitted without even noticing. Moreover, particularly in the case where a nucleic acid array in which the types of probes are narrowed is prepared, the level of importance of every probe is higher compared to a nucleic acid array on which a wide variety of probes are immobilized in an all-encompassing manner. Therefore, when statistically treating and interpreting the data of every probe obtained from the entire nucleic acid array, there is a high possibility that it will lead to radically wrong conclusions.